Outer membrane vesicles-transmitted virulence genes mediate the emergence of new antimicrobial-resistant hypervirulent Klebsiella pneumoniae

Figures & data

Figure 1. Purification and characterization of OMVs derived from hvKp and cKp strains. (A) NTA of hvKp-OMVs and (C) cKp-OMVs (n = 3). (B) TEM of hvKp-OMVs (arrows) and (D) cKp-OMVs (arrows), scale bar = 100 nm.

Figure 2. Protein and DNA concentration of the OMVs. (A) DNA concentration of hvKp-OMVs and cKp-OMVs (n = 3). (B) Protein concentration of hvKp-OMVs and cKp-OMVs (n = 3). (C) PCR profile of the hvKp-OMVs and hvKp strains used in this study. (D) Cellular localization of protein from hvKp-OMVs. (E) Biological Process (left), Cellular Component (middle) and Molecular Function (right) of protein from hvKp-OMVs. (F) Bioinformatics analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG) and (G) Protein-Protein Interaction Networks (PPI) were performed on protein from hvKp-OMVs.

Figure 3. Agarose gel electrophoresis showed REP-PCR products with expected images. REP-PCR profile of the hvKp, cKp-TC1, cKp-TC2 and cKp strains used in this study. The primers used to perform repetitive extragenic palindromic PCR (REP-PCR) are REP (left) and ERIC (right), respectively.

Table 2. Phenotypic and genotypic characteristics of donor, transformant, and recipient strains.

Strain	MIC(μg ml^−1)							prmpA
	Cefazolin	Cefotaxime	Imipenem	Meropenem	Ampicillin	Aztreonam	Tetracycline
cKp	16	2	2	<1	>16	>16	>8	–
cKp-TC1	16	2	2	<1	>16	>16	>8	+
cKp-TC2	16	2	2	<1	>16	>16	>8	+
hvKp	≤4	≤1	≤1	<1	16	≤2	≤2	+

Figure 4. hvKp-OMVs enhanced mucoviscosity and capsule production of cKp strains. (A) String tests on blood agar plates of hvKp, (B) cKp-TC1, (C) cKp-TC2, (D) cKp strains. (E) Mucoviscosity and (F) uronic acid production of hvKp, cKp-TC1, cKp-TC2 and cKp strains (+OMVs_hvKp-100 μg). Results are presented as mean ± SEM, ***P < 0.001. (G) hvKp-OMVs could induce an increased level of mucoviscosity in cKp strains (+OMVs_hvKp-500 μg), while the level of mucoviscosity in cKp strains is not enhanced by E.coli-OMVs (+OMVs_E.coli-500 μg) and cKp-OMVs (+OMVs_cKp-500 μg). Wilcoxon paired t-test, ***P < 0.001, significant difference. Wilcoxon paired t-test, ns P > 0.05, no significant difference. (H) cKp-OMVs (+OMVs_cKp-500 μg) could not induce the increased level of mucoviscosity in hvKp strains, while the level of mucoviscosity in hvKp strains is not enhanced by its own OMVs even at higher concentrations (+OMVs_hvKp-500 μg). Wilcoxon paired t-test, ns P > 0.05, no significant difference. (I)(J) PCR amplified fragments of the hvKp, cKp-TC1, cKp-TC2 and cKp strains, respectively.

Figure 5. Virulence potential of hvKp, cKp-TC1, cKp-TC2 and cKp strains with an inoculum of 5 × 10⁵ CFU at 120 h after infection in a mouse lethality assay (n = 5). The log-rank Mantel–Cox test was used for the analysis of survival curves.

Figure 6. Transformants exhibited stable hypermucoviscosity phenotype after several generations. hvKp-OMVs enhanced mucoviscosity of cKp strains. (A) String tests on blood agar plates of five generations of cKp-TC1 and cKp-TC2. (B)Mucoviscosity of five generations of cKp-TC1 and (C)cKp-TC2. (+OMV_ShvKp-100 μg). Results are presented as mean ± SEM, ***P <0.001. (D) PCR amplified fragments of five generations of cKp-TC1 and (E) cKp-TC2 (prmpA).

Table 1. Oligonucleotides used in this work.

Primer	Sequence (5′ to 3′)	Length (bp)	Use(s)
prmpA Forw	ACTGGGCTACCTCTGCTTCA	536	PCR
prmpA Rev	CTTGCATGAGCCATCTTTCA
iroB Forw	CCCTGGCATATCAAAGGCGT	534	PCR
iroB Rev	GACAACAACGCGGGCATTTA
peg-344 Forw	TGGGGTTATTCTTTCGCT	508	PCR
peg-344 Rev	TTTCCAAGCTTACTGCAATT
prmpA2 Forw	GTGCAATAAGGATGTTACATTA	430	PCR
prmpA2 Rev	GGATGCCCTCCTCCTG
iucA Forw	AATCAATGGCTATTCCCGCTG	239	PCR
iucA Rev	CGCTTCACTTCTTTCACTGACAGG
REP Forw	NNNNCGNCGNCATCNGGC	–	REP-PCR
REP Rev	NCGNCTTATCNGGCCTAC
ERIC Forw	ATGTAAGCTCCTGGGGATTCAC	–	REP-PCR
ERIC Rev	AAGTAAGTGACTGGGGTGAGCG